Nucleic acid-based compounds and methods of use thereof

ABSTRACT

The invention provides compounds capable of treating against hepatitis infections, particularly hepatitis B viral infections. Compounds of the invention are nucleic acid-based and preferably comprise 2, 3, 4, 5 or 6 nucleoside units.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/146,175, filed May 15, 2002, which claims the benefit of U.S.Provisional Application No. 60/291,471, filed on May 16, 2001. Theentire teachings of the above applications are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to nucleic-acid based compounds. Compoundsof the invention are useful for a variety of therapeutic applications,including treatment against hepatitis B virus.

BACKGROUND

Hepatitis B virus (HBV) is a compact, enveloped DNA virus belonging tothe Hepadnavirus family. The virus is a major cause of chronic liverdisease and hepatocellular carcinoma worldwide (Hoofnagle (1990) N. Eng.J. Med., 323:337-339). HBV is associated with acute and chronichepatitis and hepatocellular carcinoma and may be a cofactor in thedevelopment of acquired immune deficiency syndrome (Dinestag et al. inHarrison's Principles of Internal Medicine, 13^(th) Ed. (Isselbacher etal. eds.) McGrw-Hill, NY, NY (1993) pp. 1458-1483). At least 400 millionpeople are currently infected with HBV.

Current clinic agents, however, do not provide effective therapy or cureof HBV infections. Antiviral therapy with interferon has been used forchronic hepatitis, but has met with only partial success, and therecomplications from such therapy. Short term therapy with glucocorticoidsmay be beneficial in conjunction with interferon therapy, but long termtreatment is limited by toxicological problems (Dinestag et al., supra).

It thus would be desirable to have new agents for treatment of viralinfections. It would be particularly desirable to have new agents fortreatment against hepatitis B viral infections.

SUMMARY OF THE INVENTION

We have now found compounds and compositions that are particularlyuseful for treatment of viral infections. Preferred compounds of theinvention that can exhibit significant activity against hepatitis Bvirus.

Compounds of the invention are nucleic-based small molecules thatcontain at least two nucleoside units, and typically contain no morethan about 2, 3, 4, 5 or 6 nucleotide units, preferably no more thanabout 2, 3, or 4 nucleotide units, still more preferably a total of 2 or3 nucleoside units. Compounds of the invention comprise at least onenucleotide unit that contains one or more modifications from “natural”nucleic acids. Preferred compounds of the invention include those thathave phosphorothioate and/or phophoramidate internucleotide linkages.

Especially preferred compounds of the invention include the followingcompounds 1, 2 and 3, and pharmaceutically acceptable salts of suchcompounds:

The invention also includes therapeutic methods comprising use of one ormore compounds of the invention. Methods of the invention includetreatment of HBV infections, including treatment and prevention(prophylactic therapy) of HBV-associated disorders or diseases.

Preferred methods of the invention include administering a therapeuticeffective amount of a compound of the invention to a viral infectedcell, particularly a human cell, especially a cell that is infected withHBV. Methods of the invention also comprise administering to a mammal,particularly a primate such as a human, an effective amount of one ormore compounds of the invention.

The invention further provides pharmaceutical compositions that compriseone or more compound of the invention and a pharmaceutically acceptablecarrier.

Other aspects of the invention are disclosed infra.

DETAILED DESCRIPTION OF THE INVENTION

As discussed above, we have discovered new synthetic oligonucleotides(at least two nucleotide units) that can exhibit significant anti-HBVactivity.

Compounds of the invention comprise one or more modifications from“natural” nucleic acids, i.e. natural internucleoside linkages, bases ofG, C, T and U, etc. Modifications include, for example, modifications ofthe internucleotide linkage, the base or the sugar moiety, etc.

Compounds of the invention suitably contain two or moredeoxyribonucleotide and/or ribonucleotide monomers connected byinternucleotide linkages. Compounds of the invention may be constructedentirely of deoxyribonucleotides, entirely of ribonucleotides or of acombination of deoxyribonucleotides and ribonucleotides, includinghybrid and inverted hybrid compounds. Hybrid compounds contain a coreregion (e.g. 1, 2 or 3 units) of deoxyribonucleotides interposed betweenflanking regions of ribonucleotides (e.g. 1, 2 or 3 units). Invertedhybrids contain a core region of ribonucleotides (e.g. 1, 2, or 3 units)interposed between flanking regions of deoxyribonucleotides (e.g. 1, 2or 3 units).

Nucleotide units of compounds of the invention may be connected bystandard phosphodiester internucleotide linkages between the 5′ group ofone mononucleotide pentose ring and the 3′ group of an adjacentmononucleotide. Such linkages could also be established using differentsites of connection, including 5′ to 5′, 3′ to 3′, 2′ to 5′ and 2′ to2′, or any combination thereof. In addition to phosphodiester linkages,the mononucleotides may also be connected by alkylphosphonate,phosphorothioate, phosphorodithioate, alkylphosphonothioate,phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate,or carboxymethyl ester linkages, or any combination thereof. Asdiscussed above, preferred compounds of the invention include at leastone phosphorothioate and/or phosphoramidate linkage.

Compounds of the invention may be constructed such that allmononucleotides units (e.g. 2, 3, 4, 5 or 6 total units) are connectedby the same type of internucleotide linkages or by combinations ofdifferent internucleotide linkages, including chimeric or invertedchimeric oligonucleotides. Chimeric compounds of the invention have aphosphorothioate core region interposed between methylphosphonate orphosphoramidate flanking regions. Inverted chimeric compounds of theinvention have a nonionic core region (e.g. alkylphosphonate and/orphosphoramidate and/or phosphotriester internucleoside linkage)interposed between phosphorothioate flanking regions.

Compounds of the invention also may be constructed of adenine, cytosine,guanine, inosine, thymidine or uracil mononucleotides. Preferredcompounds of the invention may be constructed from mononucleotide unitswhich contain modifications to the base and/or sugar moiety of themononucleotide. Modifications to the base or sugar include covalentlyattached substituents of alkyl, carbocyclic aryl, heteroaromatic orheteroalicyclic groups having from 1 to 3 separate or fused rings and 1to 3 N, O or S atoms, or a heterocyclic structure.

Alkyl groups preferably contain from 1 to about 18 carbon atoms, morepreferably from 1 to about 12 carbon atoms and most preferably from 1 toabout 6 carbon atoms. Specific examples of alkyl groups include, forexample, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,pentyl, hexyl, heptyl, octyl, nonyl, decyl etc. Aralkyl groups includethe above-listed alkyl groups substituted by a carbocyclic aryl grouphaving 6 or more carbons, for example, phenyl, naphthyl, phenanthryl,anthracyl, etc.

Cycloalkyl groups preferably have from 3 to about 8 ring carbon atoms,e.g. cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, 1,4-methylenecyclohexane, adamantyl, cyclopentylmethyl, cyclohexylmethyl,1- or 2-cyclohexylethyl and 1-, 2- or 3-cyclohexylpropyl, etc.

Exemplary heteroaromatic and heteroalicyclic group include pyridyl,pyrazinyl, pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl,imidazolyl, indolyl, benzothiazolyl, tetrahydrofuranyl,tetrahydropyranyl, piperidinyl, morpholino and pyrrolidinyl.

Preferred modifications to the sugar group include modifications to the2′ position of the ribose moiety which include but are not limited to2′—O-substituted with an —O-lower alkyl group containing 1-6 saturatedor unsaturated carbon atoms, or with an —O-aryl, or allyl group having2-6 carbon atoms wherein such —O-alkyl, aryl or allyl group may beunsubstituted or may be substituted (e.g., with halogen, hydroxy,trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxy,carbalkoxyl, or amino groups), or wherein the 2′—O-group is substitutedby an amino, or halogen group. None of these substitutions are intendedto exclude the native 2′-hydroxyl group in case of ribose or 2′—H— inthe case of deoxyribose.

Preferred compounds of the invention having modified nucleotide unitsinclude 2′—O-methyl ribonucleotides (2′-OMe) and 5-methylateddeoxycytosine (5-Me-dC). Particularly preferred compounds of theinvention comprises at least one, preferably one to five 2′—O-methylribonucleotides at the 3′ end of the oligonucleotide. Moreover, acompound of the invention may further comprise at least one, preferablyone to five 2′—O-methyl ribonucleotides at the 5′-end.

Sugar groups of mononucleotide units of compounds of the invention maybe natural or modified (e.g. synthetic) and in an open chain or ringform. Sugar groups may be comprised of mono-, di-, oligo- orpoly-saccharides wherein each monosaccharide unit comprises from 3 toabout 8 carbons, preferably from 3 to about 6 carbons, containingpolyhydroxy groups or polyhydroxy and amino groups. Non-limitingexamples include glycerol, ribose, fructose, glucose, glucosamine,mannose, galactose, maltose, cellobiose, sucrose, starch, amylose,amylopectin, glycogen and cellulose. The hydroxyl and amino groups arepresent as free or protected groups containing e.g. hydrogens and/orhalogens. Preferred protecting groups include acetonide, t-butoxycarbonyl groups, etc. Monosaccharide sugar groups may be of the L or Dconfiguration and a cyclic monosaccharide unit may contain a 5 or 6membered ring of the α or β conformation. Disaccharides may be comprisedof two identical or two dissimilar monosaccharide units.Oligosacchrrides may be comprised of from 2 to 10 monosaccharides andmay be homopolymers, heteropolymers or cyclic polysugars.Polysaccharides may be homoglycans or heteroglycans and may be branchedor unbranched polymeric chains.

The di-, oligo- and poly-saccharides may be comprised of 1

4, 1

6 or a mixture of 1

4 and 1

6 linkages. The sugar moiety may be attached to the link group throughany of the hydroxyl or amino groups of the carbohydrate.

Other modifications include those which are internal or are at theend(s) of a compound of the invention and include additions to themolecule at the internucleoside phosphate linkages, such as cholesterol,cholesterol or diamine compounds with varying numbers of carbon residuesbetween the two amino groups, and terminal ribose, deoxyribose andphosphate modifications which cleave, or crosslink to the oppositechains or to associated enzymes or other proteins which bind to theviral genome. Additional linkers including non-nucleoside linkersinclude, but are not limited to, polyethylene glycol of varying lengths,e.g., triethylene glycol, monoethylene glycol, hexaethylene glycol, (Maet al. (1993) Nucleic Acids Res. 21: 2585-2589; Benseler et al. (1993)J. Am. Chem. Soc. 115: 8483-8484), hexylamine, and stilbene (Letsingeret al, (1995) J. Am. Chem. Soc. 117: 7323-7328) or any othercommercially available linker including abasic linkers or commerciallyavailable asymmetric and symmetric linkers (CloneTech, Palo Alto,Calif.) (e.g., Glen Research Product Catalog, Sterling, VA).

Additionally compounds of the invention capped with ribose at the 3′ endof the oligonucleotide may be subjected to NaIO₄ oxidation/reductiveamination. Amination may include but is not limited to the followingmoieties, spermine, spermidine, Tris(2-aminoethyl) amine (TAEA), DOPE,long chain alkyl amines, crownethers, coenzyme A, NAD, sugars, peptides,dendrimers.

Compounds of the invention also may be capped with a bulky substituentat their 3′ and/or 5′ end(s), or have a substitution in one or bothnonbridging oxygens per nucleotide. Such modifications can be at some orall of the internucleoside linkages, as well as at either or both endsof the oligonucleotide and/or in the interior of the molecule (reviewedin Agrawal et al. (1992) Trends Biotechnol. 10: 152-158). Somenon-limited examples of capped species include 3′—O-methyl, 5′—O-methyl,2′—O-methyl, and any combination thereof.

Specifically preferred compounds of the invention include the followingcompounds 1 through 62:

Compounds of the can be prepared by art recognized methods. For example,nucleotide units can be covalently linked using art-recognizedtechniques such as phosphoramidite, H-phosphonate chemistry, ormethylphosphoramidite chemistry (see, e.g., Goodchild (1990)Bioconjugate Chem. 2: 165-187; Uhlmann et al. (1990) Chem. Rev. 90:543-584; Caruthers et al. (1987) Meth. Enzymol. 154: 287-313; U.S. Pat.No. 5,149,798) which can be carried out manually or by an automatedsynthesizer and then processed (reviewed in Agrawal et al. (1992) TrendsBiotechnol. 10: 152-158). Compounds of the invention withphosphorothioate linkages can be prepared using methods well known inthe field such as phosphoramidite (see, e.g., Agrawal et al. (1988)Proc. Natl. Acad. Sci. (USA) 85: 7079-7083) or H-phosphonate (see, e.g.,Froehler (1986) Tetrahedron Lett. 27: 5575-5578) chemistry. Thesynthetic methods described in Bergot et al. (J. Chromatog. (1992) 559:35-42) can also be used. Oligonucleotides with other types of modifiedinternucleotide linkages can be prepared according to known methods(see, e.g., Goodchild (1990) Bioconjugate Chem. 2: 165-187; Agrawal etal. (1988) Proc. Natl. Acad. Sci. (USA) 85: 7079-7083; Uhlmann et al.(1990) Chem. Rev. 90: 534-583; and Agrawal et al. (1992) TrendsBiotechnol 10: 152-158).

As discussed above, the invention provides a pharmaceutical compositionwhich comprises at least one compound of the invention, preferablytogether with a pharmaceutically acceptable carrier. Specificembodiments include a therapeutic amount of a lipid carrier.

As discussed above, compounds of the invention are suitable for use in avariety of therapeutic application, particularly to treat against aviral infection in mammalian cells, particularly human cells. Compoundsof the invention are especially for use in the control or prevention ofhepatisis viruses, particularly control or prevention of hepatitis Bviral infections in human cells.

Compounds of the invention also are useful to treat againstdrug-resistant viral strains, including strains of hepatitis B virusthat are resistant to current therapies.

Preferred therapeutic methods of the invention include administering atherapeutic amount of a pharmaceutical composition containing one ormore compounds of the invention to a cell to thereby inhibit orotherwise treat against a hepatitis B viral infection. In a similaraspect, compounds of the invention can be used for treating hepatitis Bviral infections comprising the step of administering to an infectedcell or animal, particularly a primate such as a human, a therapeuticamount of a pharmaceutical composition containing at least one compoundsof the invention.

Compounds of the invention may be used in therapy in conjunction withother medicaments such as reverse transcriptase inhibitors such as adideoxynucleoside e.g. 3TC.

Also preferred is administration of multiple, distinct compounds of theinvention to a subject as part of a coordinated therapeutic regime.Particularly preferred is where at least two or more preferably allthree of compounds 1, 2 and 3 are administered to a patient as part of acoordinated administration regime.

Such combination therapy, i.e. of a compound of the invention eitherwith a distinct agent such as 3Tc, or with an additional compound of theinvention may be accomplished by administration of the same or differentpharmaceutical formulations, or sequential administration of thedistinct agents. Generally preferred however is the substantiallysimultaneous of multiple distinct agents to a patient, e.g. in a unitarypharmaceutical composition containing the compounds.

Administration of compounds of the invention may be made by a variety ofsuitable routes including oral, topical (including transdermal, buccalor sublingal), nasal and parenteral (including intraperitoneal,subcutaneous, intravenous, intradermal or intramuscular injection) withoral or parenteral being generally preferred. It also will beappreciated that the preferred method of administration and dosageamount may vary with, for example, the condition and age of therecipient.

Compounds of the invention may be used in therapy in conjunction withother phanrmaceutically active medicaments, such as another anti-viralagent, or an anti-cancer agent. Additionally, while one or morecompounds of the invention may be administered alone, they also may bepresent as part of a pharmaceutical composition in mixture withconventional excipient, i.e., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, oral or otherdesired administration and which do not deleteriously react with theactive compounds and are not deleterious to the recipient thereof.Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions, alcohol, vegetable oils, polyethyleneglycols, gelatin, lactose, amylose, magnesium stearate, talc, silicicacid, viscous paraffin, perfume oil, fatty acid monoglycerides anddiglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose,polyvinylpyrrolidone, etc. The pharmaceutical preparations can besterilized and if desired mixed with auxiliary agents, e.g., lubricants,preservatives, stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, colorings, flavorings and/oraromatic substances and the like which do not deleteriously react withthe active compounds.

For parenteral application, particularly suitable are solutions,preferably oily or aqueous solutions as well as suspensions, emulsions,or implants, including suppositories. Ampules are convenient unitdosages.

For enteral application, particularly suitable are tablets, dragees orcapsules having talc and/or carbohydrate carrier binder or the like, thecarrier preferably being lactose and/or corn starch and/or potatostarch. A syrup, elixir or the like can be used wherein a sweetenedvehicle is employed. Sustained release compositions can be formulatedincluding those wherein the active component is protected withdifferentially degradable coatings, e.g., by microencapsulation,multiple coatings, etc.

Therapeutic compounds of the invention also may be incorporated intoliposomes. The incorporation can be carried out according to knownliposome preparation procedures, e.g. sonication and extrusion. Suitableconventional methods of liposome preparation are also disclosed in e.g.A.D. Bangham et al., J. Mol. Biol., 23: 238-252 (1965); F. Olson et al.,Biochim. Biophys. Acta, 557: 9-23 (1979); F. Szoka et al., Proc. Nat.Acad. Sci., 75:4194-4198 (1978); S. Kim et al., Biochim. Biophys. Acta,728: 339-348 (1983); and Mayer et al., Biochim. Biophys. Acta, 858:161-168 (1986).

It will be appreciated that the actual preferred amounts of activecompounds used in a given therapy will vary according to the specificcompound being utilized, the particular compositions formulated, themode of application, the particular site of administration, etc. Optimaladministration rates for a given protocol of admistration can be readilyascertained by those skilled in the art using conventional dosagedetermination tests.

All documents mentioned herein are incorporated herein by reference.

The present invention is further illustrated by the following examples.These examples are provided to aid in the understanding of the inventionand are not to be construed as limitations thereof.

Example 1 Synthesis of Compounds of the Invention

Compounds of the invention can be synthesized using standardphosphoramidite chemistry (Beaucage (1993) Meth. Mol. Biol. 20: 33-61)on either an ABI 394 DNA/RNA synthesizer (Perkin-Elmer, Foster City,Calif.), a Pharmacia Gene Assembler Plus (Pharmacia, Uppsala, Sweden) ora Gene Assembler Special (Pharmacia, Uppsala, Sweden) using themanufacturers' standard protocols and custom methods.

Example 2 Biological Testing

HBV-infected human cells (2.2.15 cells) infected were treated (in vitro)with compounds of the invention. Significant depressions (greater than2-fold relative to the control) in extracellular (virion) HBV DNA levelsproduced by the cells were observed for the above compounds, includingcompounds 1, 2 and 3.

The invention has been descrbed in detail with reference to preferredembodiments thereof. However, it will be appreciated that those skilledin the art, upon consideration of this disclosure, may makemodifications and improvements within the spirit and scope of theinvention as set forth in the following claims.

1-25. (canceled)
 26. A compound having the structure of any one ofcompounds 1, 2, 4 or 7-62, or a pharmaceutically acceptable saltthereof.
 27. A pharmaceutical composition comprising the compound ofclaim 26 in combination with a pharmaceutically acceptable carrier. 28.The pharmaceutical composition of claim 27 wherein the pharmaceuticallyacceptable carrier comprises a lipid.
 29. A pharmaceutical compositionfor treating a viral infection comprising a therapeutically effectiveamount of a compound of claim 26 in combination with a pharmaceuticallyacceptable carrier.
 30. A pharmaceutical composition for treating an HBVinfection comprising a therapeutically effective amount of a compound ofclaim 26 in combination with a pharmaceutically acceptable carrier. 31.A pharmaceutical composition for treating a viral infection comprising atherapeutically effective amount of a compound of claim 26 incombination with a therapeutically effective amount of a reversetranscriptase inhibitor and a pharmaceutically acceptable carrier.
 32. Apharmaceutical composition of claim 31 wherein the reverse transcriptaseinhibitor is 3TC.
 33. A pharmaceutical composition of claim 30 whereinthe HBV infection is a drug resistant HBV infection.